Enzyme Activity Measurements Using Bio-Layer Interferometry

ABSTRACT

Disclosed are enzyme assays using biolayer interferometry. Assays may be carried out using immobilized substrate or with a substrate capture format. In certain embodiments, the assays are carried out using unlabeled substrates. The methods are broadly applicable to enzyme assay measurements, can be carried out in vivo or in vitro, and are easily multiplexed.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of and claims priority from U.S.application Ser. No. 11/326,689, filed Jan. 6, 2006, which isincorporated by reference in its entirety for all purposes, and furtherclaims priority from both U.S. Provisional Application Ser. No.60/645,153, filed Jan. 19, 2005 and U.S. Provisional Application Ser.No. 60/642,454, filed Jan. 7, 2005, both of which are incorporatedherein by reference in their entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to interferometry-based methods and compositionsuseful for measuring enzyme activity.

2. Description of the Related Art

Enzymes represent a broad class of proteins that catalyze biochemicalreactions and have many therapeutic and industrial applications. Oftenin the course of development and manufacture of enzyme products it isnecessary to measure the activity of the enzyme. A simple enzymeactivity method, preferably label free thereby avoiding perturbation ofthe enzyme/substrate interaction, would find wide application. In thedevelopment and manufacture of enzyme or enzyme inhibitor based productsfor therapeutic or industrial applications, it is critical to monitorthe activity of the enzyme throughout the process. Enzyme assaystypically require labeling the substrate in such a way that the enzymeacting on the substrate produces a detectable change in signal. Labeledenzyme substrates are often not commercially available, in which casetheir synthesis can be complex. For companies developing a multitude ofenzyme products, the implementation of activity methods that are simpleand easy to perform in research & development and manufacturingenvironments becomes a major task. The need for labeled substrates addsto the time, expense and inconvenience of enzyme activity measurements.Specific activity measurements also require quantifying the amount ofenzyme present in a sample. Quantitation, as by, e.g., enzyme-linkedimmunosorbant (ELISA)-based assays are also adds to the time and expenseof specific activity measurements and requires additional sample. Thepresent invention addresses these and other shortcomings of the priorart by providing simple, fiber based, real-time enzyme activity assays,capable of providing specific activity measurements, suitable forlow-volume samples, that are highly multiplexable and in someembodiments can be carried out using unlabeled substrates.

SUMMARY OF THE INVENTION

The present invention is defined by the following claims, and nothing inthis section should be taken as a limitation on those claims. Disclosedherein are assemblies, kits, and methods for assaying enzyme activityusing fiber-based interferometry. In one embodiment, the assay comprisesproviding an optical element coupled to a light source via an opticalfiber and the element includes proximal and distal reflecting surfacesseparated by at least 50 nm. A layer of enzyme substrate molecules ispositioned so that interference between beams reflected from theproximal and distal reflecting surfaces varies as an enzyme reacts withthe substrate. The reflected beams are coupled into the optical fiber.The element is exposed to an enzyme and a change is detected in theinterference between the reflected beams. The detected change isindicative of enzyme activity.

In still another embodiment, a layer of analyte binding moleculessubstituted in the optical element for the layer of enzyme substratemolecules. Interference between beams reflected from the proximal anddistal reflecting surfaces varies as an enzyme reacts with the substrateand the acted-upon substrate or portion thereof binds to the analytebinding molecules. In preferred embodiments, the analyte bindingmolecules comprise an antibody, an antibody fragment, a single chain Fvmolecule (“scFv”), an avidin, a streptavidin, or a biotin.

In another embodiment, a semi-permeable membrane is placed between theoptical element and the assay solution. In another embodiment, thesubstrate is coupled to a support such as a microtitre well or a bead.

In yet another embodiment, a similar, second element is provided thatincludes a layer of molecules that specifically binds to the enzyme. Thesecond element is exposed to an enzyme (either at the same time or at adifferent time as the first element is exposed) and a change is detectedin the interference between the reflected beams. The change isindicative of enzyme concentration or amount. This is useful forcarrying out specific activity measurements. In preferred embodiments,the enzyme-binding molecules comprise an anti-enzyme antibody, anantibody fragment or an scFv molecule.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, and accompanying drawings, where:

FIG. 1 is a graph illustrating minimum molecular size detection;

FIG. 2 is another graph illustrating minimum molecular size detection;

FIG. 3 is a graph illustrating subtilisin activity measurement at threeenzyme concentrations;

FIG. 4 is a graph illustrating subtilisin activity measurement at 50μg/ml;

FIG. 5 is a graph illustrating effect of protease inhibitor onsubtilisin activity;

FIG. 6 is a second graph illustrating effect of protease inhibitor onsubtilisin activity.

FIG. 7 is a diagram illustrating substrate preparation for substratecapture format; FIG. 7A illustrates substrate having multiple proteasecleavage sites; FIG. 7B illustrates substrate having single (or few)cleavage sites;

FIG. 8. is a schematic illustrating principle of substrate captureformat assays;

FIG. 9 is a schematic illustrating method to detect nucleotidetransferase activity;

FIG. 10 is a schematic illustrating method to detect nucleotidetransferase activity with hapten incorporation;

FIG. 11 is a schematic illustrating method to detect phosphotransferaseactivity using antibody to phosphorylated substrate;

FIG. 12 is a schematic illustrating method to detect protein kinase Cactivity;

FIG. 13 is a schematic illustrating method to detect activation ofmitogen activated protein kinase (MAPK).

DETAILED DESCRIPTION OF THE INVENTION Advantages and Utility

Briefly, and as described in more detail below, described herein areassemblies, kits and methods for assaying enzyme activity usingfiber-based interferometry.

Several features of the current approach should be noted. Measurementscan be carried out using extremely small sample volumes (e.g., nL).Measurements can be carried out in vivo or in vitro. In someembodiments, measurements can be carried out on unlabeled substrateswhile in other embodiments, substrates include a moiety to allow captureby the assembly. In preferred embodiments, the moiety is one member of abinding pair such as, e.g., avidin, streptavidin, biotin, a hapten, anantibody, antibody fragment, an scFv, or a lectin, and the opticalelement comprises the complementary member of the pair. In theseembodiments, the same type of optical element (i.e., carrying one memberof the binding pair) can be used in a wide variety of enzyme assaysprovided the substrate includes the other member of the pair.

Advantages of this approach are numerous. Because the invention providesfor fiber-based interferometry measurements, it is sensitive, capable ofbeing highly multiplexed, and easily adapted for specific activitymeasurements by including a module for measuring enzyme amount.

The invention is useful for measuring enzyme activity in any context forwhich such activity measurements are useful including, e.g., fordiscovery, modification, optimization, production, etc. of enzymes orenzyme inhibitors. The invention may be practiced using any type ofenzyme such as, e.g., hydrolases, glycosylases, esterases, andtransferases, or inhibitors of such enzymes.

DEFINITIONS

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

The term “in vivo” refers to processes that occur in a living organism.

Abbreviations used in this application include the following:dsDNA—double-stranded DNA; dNTPs—deoxynucleotide triphosphates;B-ATP—biotinylated-ATP; PEG—polyethylene glycol,PMSF—phenylmethylsulfonyl fluoride.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise.

Assemblies of the Invention

Assemblies of the invention include biosensor tips adapted for couplingto a bio-layer interferometer and carrying a layer of substrate or ananalyte binding molecule. The analyte binding molecule may be by way ofexample and not limitation, a member of a binding pair such as, e.g.,avidin, streptavidin, biotin, a hapten, an antibody, an antibodyfragment, an scFv, or a lectin.

Kits of the Invention

Kits of the invention include a glass fiber adapted for coupling to abio-layer interferometer, reagents and instructions for derivatizing theglass fiber with a substrate layer or an analyte binding molecularlayer, optionally reagents and instructions for optically activating anend of the glass fiber and packaging.

Methods of the Invention

In general, methods of the invention are practiced using assemblies andapparatus, including a bio-layer interferometry (BLI) sensor, such asthose described in co-owned U.S. Non-provisional application Ser. No.10/981,901, filed Nov. 4, 2004, for Hong Tan, et al., entitled“Fiber-Optic Assay Apparatus Based on Phase-Shift Interferometry,”Attorney Docket No. 24377-09611US, the contents of which are hereinincorporated by reference in their entirety.

In brief, the sensor is prepared by optically activating one end of aglass fiber. The activation steps include buffing the fiber surface,coating the surface with Ta₂O₅ followed by coating with an SiO₂ layer,and cleaning, and immobilization of enzyme substrate or one member of abinding pair by passive adsorption and/or covalent attachment.

Included within the scope of the invention are two broad and generalformats for assaying enzyme activity. In the first format, substrate isimmobilized on a surface of a bio-layer interferometry (BLI) sensor. Inthe second format, the BLI sensor includes a surface having a moleculecapable of binding substrate. In the second format, a semi-permeablemembrane optionally is included between the BLI sensor and the assaysolution, or the substrate is bound to a support such as a microtitrewell or a bead. These embodiments are particularly useful withhydrolases to prevent or slow binding of full-length substrate to theBLI sensor. In the second format, information about enzyme activity canbe derived both from kinetic and steady state components of theinterference signal.

A Bio-Layer Interferometry (BLI) sensor is capable of measuring subnanometer changes in the thickness of its optical layer detectionsurface. Analysis of biological samples is possible by designing assayformats where biomolecules bind at the sensor surface and change theoptical layer thickness. The magnitude of the optical layer thicknesschange is proportional to the mass or molecular weight of the bindingmolecule. The Bio-Layer Interferometer can be configured to havesubstrate immobilized to the sensor surface to measure enzymes whoseactivity creates a change in the substrate molecular weight, eitherincreasing or diminishing the molecular weight, to produce acorresponding change in the optical layer thickness.

The invention is broadly applicable to enzyme activity measurements,including by way of example but not limitation, measurements ofhydrolases (including proteases), glycosylases, esterases, transferases(including nucleotide transferases and phosphotransferases). These areconsidered in greater detail below.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for. Unless otherwise specified,procedures are carried out at room temperature (typically 20-23 degreesCelsius).

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed.(Plenum Press) Vols A and B(1992).

Example 1 BLI Molecular Weight Detection Characterization

The minimum molecular weight of a binding molecule that BLI can detectis illustrated in FIGS. 1 & 2. In FIG. 1 data of a biotin-PEG conjugatewith a molecular weight of 900 Daltons binding to a streptavidin coatedBLI sensor is depicted. The BLI sensor and methods for coating thesensor are described in detail in co-owned U.S. Non-provisionalapplication Ser. No. 10/981,901, filed Nov. 4, 2004, for Hong Tan, etal., entitled “Fiber-Optic Assay Apparatus Based on Phase-ShiftInterferometry,” Attorney Docket No. 24377-09611US. FIG. 2 shows thebinding of biotin (M.W. ˜230 D) to a streptavidin coated BLI sensor. Thedata indicate that the described interferometry methods readily detectbinding of molecules around 250 Dalton molecular weight, and thatmolecules in the 500 to 1000 Dalton molecular weight range generate asubstantial change in optical layer thickness. Based on the minimummolecular size detection of BLI, one can configure the BLI sensor withimmobilized substrates to monitor the activity of enzymes producingmolecular size changes in the substrate as small as 250 to 1000 Daltons.

The small minimum molecular size detection limit of BLI makes itpossible to apply BLI to a large number of enzymes. The following are byway of example, but not limitation, enzyme classes whose activities canbe measured in accordance with the present invention, and specificexamples of such measurements.

Example 2 Hydrolase Activity Measurements

Hydrolyases are enzymes that catalyze cleavage of C—O, C—N, C—C orphosphoric anhydride bonds.

Subgroup 1: Proteases (Enzymes Acting on Peptide Bonds)

Immobilized Substrate Format

This format features the protease substrate immobilized to the surfaceof the BLI glass fiber sensor using methods described in co-owned U.S.Non-provisional application Ser. No. 10/981,901, filed Nov. 4, 2004, forHong Tan, et al., entitled “Fiber-Optic Assay Apparatus Based onPhase-Shift Interferometry,” Attorney Docket No. 24377-09611US(incorporated herein by reference) and below. The fiber is immersed inan enzyme-containing sample, and monitored for changes in optical layerthickness.

The basic assay protocol is to incubate a Bio-Layer Interferometer (BLI)sensor on which has been immobilized an enzyme substrate in anenzyme-containing solution enzyme. The amount of the substrate depletionis quantified by, e.g., a change in optical phase shift using theBio-Layer Interferometry (BLI) technique fully described in co-owned andpending U.S. Non-provisional application Ser. No. 10/981,901, filed Nov.4, 2004, for Hong Tan, et al., entitled “Fiber-Optic Assay ApparatusBased on Phase-Shift Interferometry,” Attorney Docket No. 24377-09611US,incorporated herein by reference. The change in, e.g., optical phaseshift is proportional to the amount of enzyme activity in the solutionbecause hydrolytic activity is estimated by measuring the depletion ofsubstrate from the BLI sensor resulting from the enzyme activity.

Subtilisin Activity Measurements on Immobilized Casein Substrate

Methods

An optical signal baseline was established by immersing theoptically-activated end of a fiber sensor tip in PBS and monitoring theoptical signal using interferometry methods and instrumentationdescribed in co-owned U.S. Non-provisional application Ser. No.10/981,901, filed Nov. 4, 2004, for Hong Tan, et al, entitled“Fiber-Optic Assay Apparatus Based on Phase-Shift Interferometry,”Attorney Docket No. 24377-09611US (incorporated herein by reference inits entirety). Next the fiber was coated with Poly-D-Lysine byincubating the tip in a 0.5 mg/mL Poly-D-Lysine solution (in PBS, pH7.4) for 15 minutes. Unbound Poly-D-Lysine was rinsed by incubating thetips in PBS for 10 minutes.

The fiber was coated with a layer of casein [Sigma Chemical Company, StLouis, Mo.] by incubating the tip in a 50 μg/mL casein solution (50 mMNa Phosphate, 150 mM NaCl, pH 7) for 15 minutes. Unbound casein wasrinsed by incubating the tip in PBS for 10 minutes.

Fibers with immobilized casein were incubated in various concentrations(1, 10, 50 μg/mL) of subtilisin [Sigma Chemical Company, St Louis, Mo.]solution (in 50 mM Na Phosphate, 150 mM NaCl, pH 7). Each of theprocedures described above was carried out while monitoring the opticalsignal.

Results and Discussion

FIG. 3 illustrates the result of this example. The optical traces showcalculated illustrate biolayer thickness as a function of time. Apparentare increases in biolayer thickness during casein loading and subsequentdecreases following subtilisin incubation. The traces show a cleardose-response effect with more rapid and greater changes occurring withhigher subtilisin concentration over the tested range.

Effect of Protease Inhibitor on Enzymatic Activity

Methods

Fiber sensor tips were prepared and coated with casein as describedabove. One fiber was incubated in 50 μg/mL of subtilisin solution (in 50mM Na Phosphate, 150 mM NaCl, pH 7).

Other fibers were incubated in a pre-mixed and pre-incubated solution of50 μg/mL of subtilisin and 3 mM PMSF [Sigma Chemical Company, St Louis,Mo.] (the mixture was incubated at 37° C. for 20 minutes) solution (50mM Na Phosphate, 150 mM NaCl, pH 7). Each of the procedures describedabove was carried out while monitoring the optical signal.

Result and Discussion

Traces obtained from a fiber incubated in subtilisin solution show theexpected depletion of casein from the tip surface (FIG. 4). This showsup as smooth, time-dependent change in the trace following incubation inthe enzyme solution. Fibers incubated in the subtilisin/PMSF mixture ofsubtilisin and (protease inhibitor) PMSF did not show the anytime-dependent changes in the optical signal (FIGS. 5 and 6),illustrating inhibition of subtilisin by 3 mM PMSF.

Substrate Capture Format

The substrate capture format entails digestion of the substrate by aprotease in liquid phase followed by binding of the substrate to thesurface of the BLI sensor. The binding of the substrate is designed sothat proteolytic cleavage of the substrate produces a detectable tochange in the optical layer thickness. In one preferred embodiment, astreptavidin/biotin binding pair is used to effect substrate capture.FIG. 7 shows two approaches to tag the peptide substrate with biotin. Inthe case where the protease has multiple cleavage sites, the biotin doesnot have to be substituted at a specific site since the resultingdigestion will produce a peptide with a sufficiently small molecularsize. The substrate can also be designed so that there is a singlecleavage site and the location of the biotin substitution is such thatit remains on the smallest peptide fragment upon proteolysis. Methodsfor derivatizing substrate with biotin or another member of a bindingpair are well known to ordinarily-skilled practitioners and includebiochemical modification of existing substrate molecules, or, synthesisusing derivatized sub-units. Such methods are described in, e.g.,[Antibodies: A Laboratory Manual (E. Harlow and D. Lane, 1988);Bioconjugation Protocols: Strategies and Methods (Methods in MolecularBiology (Clifton, N.J.), V. 283, 2004)] incorporated by reference, andexemplified below.

Example 3 Trypsin Activity Measurements Using Substrate Capture

Trypsin activity measurements are made using the substrate captureformat and cytochrome C as a substrate. Cytochrome C is about 12kDaltons in molecular weight and includes has 8 trypsin cleavage sites.A standard biotin-NHS derivative [Pierce Biotechnology, Rockford Ill.]is used to tag the cytochrome C. Coupling conditions employ standardphosphate buffered saline (PBS) pH 7 buffer. Biotin-NHS is mixed withcytochrome C at a molar coupling ratio of 5 to 1 (biotin to cytochromeC) typically resulting in one biotin substitution per cytochrome Cmolecule. Streptavidin-coated BLI sensors are prepared from 0.6 mmdiameter glass fibers with a tantalum oxide layer of about 20 nm and asilicon dioxide layer of about 700 nm are dipped in a PBS solutioncontaining 0.5 mg/ml of poly-D-lysine as described above. After 15minutes at room temperature, the fibers are washed in PBS then immersedin a 1 mg/ml solution of bovine serum albumin (BSA) labeled withN-succinimidiyl 3-(2-pyridyldithio) propionate (SPDP) [PierceBiotechnology, Rockford Ill.] and incubated for 20 minutes followed by awash step with PBS. The fibers are then immersed in a solution of 50 mMdithiolthreitol for 30 minutes at room temperature. After a wash in PBS,the fibers are placed in the solution containing 20 μg/ml ofstreptavidin labeled with SMCC and incubated for 60 minutes, followed aby PBS wash. The fibers are stored in PBS until use.

The basic assay for protease activity using the substrate capture formatentails adding to a biotinylated cytochrome C solution, in the range ofabout 1 μg/ml to 1 mg/ml, a trypsin sample, typically at a 1% wt./wt.ratio. After a digestion time period, a streptavidin coated sensor isplaced in the enzyme/substrate mixture for substrate capture. To avoidtrypsin acting upon the streptavidin, the enzyme can be inactivatedeither by snap boiling the substrate mixture or adding a proteaseinhibitor, such as aprotinin, just prior to adding the glass fiber. TheBLI sensor can assess the change in substrate size by a measuring theoptical layer thickness upon binding the biotinylated peptide. FIG. 8illustrates the assay format where protease activity produces smallerpeptides with a thinner optical layer.

Subgroup 2: Glycosylases (Enzymes that Hydrolyse O or S or N GlycosylBonds)

Immobilized Substrate Format

The following example is described for dextranase, but the same methodscan be applied to other polysaccharide processing enzymes such asamylase and cellulase. Dextran is linked to a protein coated BLI sensorby initially reacting the dextran with sodium periodate to introducereactive aldehyde groups on the dextran polymer. A protein coated BLIsensor (such as prepared according to the methods described in theexample above) is then immersed in the dextran solution. The aldehydegroups on the dextran form bonds with free amino groups on the proteinresulting in a BLI sensor with a dextran as the final layer. Measurementof dextranase activity is possible when the dextran coated sensor isimmersed in a dextranase containing sample. Hydrolytic activity of thedextranase reduces the molecular size of the immobilized dextran, whichis detected as a thinner optical layer.

Substrate Capture Format

Measurement of glycosylases in the substrate capture format isaccomplished by biotinylating the polysaccharide substrate. In thedextranase example, dextran is reacted with sodium periodate to generatealdehyde groups followed by the addition of large molar excess of abis-amine, such as ethylenediamine. One amino group of the bis-aminecouples to the aldehyde on the dextran leaving the second amine free forcoupling to biotin-NHS in a subsequent reaction. The molar couplingratio of the biotin-NHS reaction is selected to yield about one biotinsubstitution per dextran. The assay for dextranase activity in thesubstrate capture format is similar to the protease second example wherethe biotinylated dextran substrate is incubated with the dextranasesample followed by binding of the biotinylated dextran to a streptavidincoated BLI sensor. Dextranase activity creates smaller dextran fragmentswhich are measured as a thinner optical layer.

Example 4 Esterase Activity Measurements

Subgroup 3: Esterases (Enzymes that Act on Ester Bonds)

Some examples of esterases include nucleases (RNase, DNase, etc.),alkaline phosphatase, acid phosphatase, and serine/threoninephosphatase. DNase I is used as an example of measuring esteraseactivity. Since DNase I cleaves at all 4 bases in oligonucleotides assmall as three bases in length, dsDNA is prepared by a commercial vendorusing standard DNA synthesis techniques having a length of about 30-40base pairs with a biotin group at one of the terminal ends. Thebiotinylated dsDNA is bound to a streptavidin coated BLI sensor. Thesensor is then immersed in sample containing DNase I in the buffer: 10mM Tris pH 7.5, 2.5 MgCl₂, 0.5 nM CaCl₂ and the decrease in opticallayer thickness is monitored. The alternative substrate capture formatis performed by mixing the biotin labeled dsDNA with the DNase I inliquid phase before the binding of the substrate to the streptavidincoated sensor.

Example 5 Transferase Activity Measurements

Transferases are enzymes that catalyze the transfer of methyl, glycosyl,or phospho groups to other compounds. In contrast to the hydrolyticenzymes, the transferases increase the molecular size of the substrateand their activity is detected by the BLI sensor as an increase inoptical layer thickness.

Subgroup 1: Nucleotide Transferases

Nucleotides transferases catalyze the incorporation of nucleotides intoDNA or RNA polymers. FIG. 9 shows the format for measuring DNApolymerase I activity as an example. A DNA template from 10 to 30nucleotides is obtained from a commercial vendor with a biotinincorporation at either of the terminal nucleotides. The DNA template ishybridized with an oligonucleotide primer enabling the DNA polymerase Ito incorporate nucleotides in the 5′ to 3′ direction. The biotin taggedDNA template can be bound to a streptavidin coated sensor either beforeor after the DNA polymerase step. Conditions for the polymerase stepsuch as enzyme loading, dNTPs, buffer formulation, etc. followestablished protocols. Since the dNTPs have molecular weights around 400D, incorporation of as little as 1-2 nucleotides can be detected as achange in optical thickness.

An alternative approach to measure activity of nucleotide transferasesis based on hapten incorporation as shown in FIG. 10. A DNA templatewith a hybridized primer as mixed with DNA polymerase I and a mixture ofdNTPs including biotinylated ATP. B-ATP is obtained from commercialsources since it is commonly used in nick translation. After thepolymerase step, a streptavidin coated BLI sensor is placed in thesample mixture. Unincorporated B-ATP having a molecular weight around600 D produces a relatively small increase in the optical layer uponbinding to streptavidin, whereas B-ATP incorporated in the DNA templateproduces a greater increase in the optical layer depending on themolecular weight of the DNA.

Subgroup 2: Phosphotransferases

Phosphotransferases catalyze the transfer of phosphate groups tohydroxyl containing compounds, typically peptides with tyrosine, serine,or threonine residues. There are over 100 commercially monoclonalantibodies that bind to a phosphorylated amino acid or specificsequences encompassing a phosphorlyated amino acid. Many assays ofkinase activity have been reported and are commercially availableutilizing antibodies to phosphorylated peptide substrates. FIG. 11describes an assay using an anti phosphotyrosine antibody. The antibodyis initially biotinylated by standard methods then bound to astreptavidin coated sensor. The substrate (Glu₄Tyr)_(n) is incubatedwith tyrosine kinase plus ATP, after which the sensor coated with antiphosphotyrosine is placed in the sample. The binding of thephosphorylated peptide produces an increase in the optical layerthickness.

FIG. 12 illustrates an assay using an antibody to a phosphorylated aminoacid sequence for a specific kinase. In this case, the kinase is proteinkinase C and commercial antibody to phosphorylated protein kinase Csubstrate. FIG. 13 shows an exemplary format for a kinase activationassay. In this example, mitogen activated protein kinase (MAPK) isactivated by phosphorylation by another enzyme, MEK1. A commercial antiphospho MAPK bound to a BLI sensor detects the activation of MAPK.

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

1. An assembly, comprising an optical element adapted for coupling to alight source via an optical fiber, the optical element including (a) aproximal reflecting surface and a distal reflecting surface separated byat least 50 nm, and (b) a layer of enzyme substrate molecules positionedso that interference between a reflected beam from the proximalreflecting surface and a reflected beam from the distal reflectingsurface varies as an enzyme reacts with the substrate.
 2. An assembly,comprising an optical element adapted for coupling to a light source viaan optical fiber, the optical element including (a) a proximalreflecting surface and a distal reflecting surface separated by at least50 nm, and (b) a layer of analyte binding molecules positioned so thatinterference between a reflected beam from the proximal reflectingsurface and a reflected beam from the distal reflecting surface variesas an enzyme reacts with the substrate.
 3. A kit, comprising a glassfiber adapted for coupling to a bio-layer interferometer, reagents andinstructions for derivatizing the glass fiber with a substrate layer oran analyte binding molecular layer.
 4. The kit of claim 3, furthercomprising reagents and instructions for optically activating an end ofthe glass fiber.